This project seeks to define the interactions between two major inputs to the myenteric cholinergic neurons, namely the neurokininergic (NK) and serotoninergic (5-HT) neurons of the guinea pig small intestine. Since both excitatory and inhibitory signals can be conveyed to the cholinergic neurons, we have four main objectives: (1) To examine how different agonists for NK and 5-HT receptors modulate the release of acetylcholine (ACh) from the myenteric cholinergic neurons. There is evidence to suggest a functional coupling between NK and 5-HT receptors on cholinergic neurons. We will determine if this interplay is restricted to any specific receptor subtype, or if it is mediated by a release of 5-HT evoked by NK, or if the action of NK on the cholinergic neurons is dependent on a subthreshold level of 5-HT. (2) To determine if eicosanoids are responsible for the modulation of ACh secretion by NK and 5-HT. Arachidonic acid (AA) from an activation of phospholipase A2, on membrane phospholipids or a metabolite converted from AA can affect ACh release. There is evidence to suggest that a part of this eicosanoid-pathway mediates both NK and 5-HT action. The exact involvement of eicosanoids will be determined by study of isolated ganglia devoid of muscle or tissue contaminants. The release of AA will be measured. The lipoxygenase, cyclooxygenase, and epoxygenase metabolic pathways involved in the conversion of AA will be examined. (3) To determine if the actions of NK and 5-HT are mediated by a specific type of voltage-sensitive Ca2+ channels (VSCC) or by an action directed at the intracellular mobilization of Ca+2 stores, or both. Experiments will determine if the IP3-sensitive Ca2+ stores are involved in the actions of NK and 5-HT. We will employ chemical probes which are selective at enhancing Ca2+ entry, or immobilizing internal stores without affecting Ca2+ entry, or do not activate membrane hydrolysis of phospholipids but deplete internal Ca2+ stores. One aspect of our study will focus on the interdependence between entry of Ca2+ and mobilization of internal Ca2+ stores of IP3. (4) To determine the role of protein kinase C (PKC) in the NK-and 5-HT- receptor activated ACh release by using affinity purified cholinergic terminals. Highly specific PKC inhibitors and activators will be used. Due to its tight association with plasma membrane, PKC could mediate transmitter release by modification of ion channels. A decisive test for this can only come from a study using purified cholinergic terminals.